Integrin-mediated Adhesion Regulates Cell Polarity and Membrane Protrusion through the Rho Family of GTPases

Abstract

Integrin-mediated adhesion is a critical regulator of cell migration. Here we demonstrate that integrin-mediated adhesion to high fibronectin concentrations induces a stop signal for cell migration by inhibiting cell polarization and protrusion. On fibronectin, the stop signal is generated through ␣5␤1 integrin-mediated signaling to the Rho family of GTPases. Specifically, Cdc42 and Rac1 activation exhibits a biphasic dependence on fibronectin concentration that parallels optimum cell polarization and protrusion. In contrast, RhoA activity increases with increasing substratum concentration. We find that cross talk between Cdc42 and Rac1 is required for substratum-stimulated protrusion, whereas RhoA activity is inhibitory. We also show that Cdc42 activity is inhibited by Rac1 activation, suggesting that Rac1 activity may down-regulate Cdc42 activity and promote the formation of stabilized rather than transient protrusion. Furthermore, expression of RhoA down-regulates Cdc42 and Rac1 activity, providing a mechanism whereby RhoA may inhibit cell polarization and protrusion. These findings implicate adhesion-dependent signaling as a mechanism to stop cell migration by regulating cell polarity and protrusion via the Rho family of GTPases. INTRODUCTIONCell migration plays a central role in both normal and pathological processes, including embryonic development, wound healing, inflammation, and tumor metastasis (Trinkaus, 1984). Integrin-mediated adhesion to the extracellular matrix is a critical regulator of cell migration speed for many cell types, including fibroblasts and carcinomas. These cell types exhibit a biphasic relationship between cell migration speed and substratum concentration (Goodman et al., 1989;DiMilla et al., 1993;Huttenlocher et al., 1996;Ho et al., 1997;Palecek et al., 1997), with maximum migration rates at intermediate adhesiveness where cells can both efficiently form adhesions at the cell front and release adhesive contacts at the cell rear (DiMilla et al., 1991;Duband et al., 1991;Huttenlocher et al., 1995). Previous studies have implicated reduced adhesive release at the cell's rear as an important mechanism for inhibited migration under conditions of high cell substratum adhesiveness (Marks et al., 1991;Hendey et al., 1992;Jay et al., 1995;Huttenlocher et al., 1997;Cox and Huttenlocher, 1998;Palecek et al., 1998).Integrins are a family of heterodimeric cell surface adhesion receptors that bind to specific extracellular matrix components and cluster in the membrane to form organized adhesive contacts called focal complexes or focal adhesions (Hynes, 1992). To migrate, cells must coordinately assemble and disassemble integrin-containing adhesive complexes. Integrin-containing adhesive complexes regulate cell migration by performing both an adhesive function, linking the extracellular matrix to the actin cytoskeleton, and a signal transduction function by regulating molecules important for cell motility (Clark and Brugge, 1995;Huttenlocher et al., 1995; Ilic et al., 1995;Cary et al., 1996Cary ...

Dendritic Fibroblasts in Three-dimensional Collagen Matrices

“…For fibroblasts on coverslips in the presence of LPA or PDGF, on the other hand, blocking Rho caused cells to form extensions that were collapsed and distorted. Small G protein activation and its consequences for cell behavior have been shown to be regulated not only by growth factors but also by cell adhe- sion (Hall, 1998;Price et al, 1998;Kaibuchi et al, 1999;Ren et al, 1999;Arthur et al, 2000;del Pozo et al, 2000;Cox et al, 2001). Our findings suggest that Rho signals generated by fibroblast adhesion on planar surfaces impact cells differently from signals generated by fibroblast adhesion in the three-dimensional collagen matrices.…”

R-Ras Controls Membrane Protrusion and Cell Migration through the Spatial Regulation of Rac and Rho

“…For example, activated Rac has been reported to decrease or increase Rho activity in different cell types (Sander et al, 1999;Zondag et al, 2000;Cox et al, 2001;Li et al, 2002). In addition, although activated Rac can lead to increased Rho activity in some cells, this newly active Rho then inhibits Rac activation, suggesting a negative feedback loop by which Rho and Rac activity are precisely regulated (Li et al, 2002).…”

“…To address the possibility that R-Ras inhibits protrusion and migration through effects on cell adhesion, we plated cells expressing activated R-Ras (38V) on various collagen concentrations. Lower ECM concentrations have been shown to enhance cell migration by reducing integrinmediated adhesion, whereas higher concentrations decrease motility (Huttenlocher et al, 1996;Palecek et al, 1997;Cox et al, 2001). However, altering collagen concentration from 0.1 to 300 g/ml could not rescue the migratory defect in R-Ras (38V)-expressing cells (Figure 1, dashed line).…”

“…Expression of dominant negative R-Ras (41A) increases directional persistence (*p Ͻ 0.05 vs. control). R-Ras enhances integrin avidity and cell adhesion (Zhang et al, 1996;Sethi et al, 1999;Kwong et al, 2003), both of which affect random cell migration (Palecek et al, 1997, Cox et al, 2001. To address the possibility that R-Ras inhibits protrusion and migration through effects on cell adhesion, we plated cells expressing activated R-Ras (38V) on various collagen concentrations.…”

Mature Dendritic Cells Express Functional Thrombin Receptors Triggering Chemotaxis and CCL18/Pulmonary and Activation-Regulated Chemokine Induction

“…4A). ROCK2 was constitutively activated in nonstimulated LPS-matured DC, which might be due to cell adhesion and engagement of integrins (30). Neither thrombin nor PAR-APs were found to enhance ROCK2 activity (Fig.…”